Sequence-specific isolation and detection of selected polynucleotides in complex biological specimens is an important process in many fields, including medical diagnostics and drug research. In spite of recent advances, the process remains relatively slow, complex, and labor intensive. In a typical method (Gillespie and Spiegelman, 1965), single-stranded polynucleotides are fixed on a suitable surface, such as a nitrocellulose filter. A labeled sequence-specific probe is then added under annealing conditions, and, following a suitable annealing period (typically many hours), unpaired probe is washed away, and the surface is assessed for label. While this strategy has proven extremely valuable in a broad range of applications, it is quite slow and labor intensive, and affords relatively low signal to noise discrimination, particularly when the sequence to be detected is present at low concentrations, as is often the case with clinical specimens.
In various solution-based strategies, the sequence-specific pairing step is carried out in solution, followed by capture of the paired probe on a suitable surface. A number of methods have been developed to increase the speed of pairing between complementary sequences. These generally serve to increase the concentration of nucleic acids in the specimen, e.g. at a phenol/water interface (Kohne et al., 1977) or by including additives which physically exclude nucleic acids from a substantial volume of the solution (Wetmur 1975; Chang et al., 1974). This increased pairing rate, however, is generally accompanied by a significant increase in background signal.
Because of these limitations, such methods remain unsuited for many commercial applications. In particular, conventional sample preparation methods for amplification-based diagnostics, such as PCR, often isolate polymerase inhibitors, which are present in many clinical samples, along with the desired polynucleotides.
It is therefore desirable to provide a method of detecting or isolating target polynucleotides from complex samples which is both rapid and selective, allows detection of low levels of analyte, and requires a minimum of complex manipulations.